Colorectal cancer (CRC) is a cancer of the large bowel and is the third most common cancer in men and women, representing 13% of all cancers. About 20% of patients have metastatic disease at the time of diagnosis, and 50% of all colorectal cancer patients will develop metastases and ultimately die from their disease. In the United States alone around 131,000 people will be diagnosed with colorectal cancer and 56,000 will die each year. The lifetime CRC risk in the general population is 5%, but this figure rises dramatically with age: by the age of 70 years, approximately half of the Western population will develop an adenoma. Nearly 85% of all CRC cases are sporadic in origin (“somatic CRC”) and the rest occur as a result of an inherited genetic mutation (“hereditary CRC”).
Colorectal cancers arise from adenomas, which are dysplastic but nonmalignant precursor lesions in the colon. Progression to carcinoma occurs through the accumulation of multiple somatic mutations, ultimately leading to malignant transformation and the formation of an invasive cancer. One of the most critical genes mutated in the progression to CRC is the Adenomatous Polyposis Coli (APC) tumor suppressor. Since APC mutations are detected very early in the adenoma-carcinoma sequence, the APC protein has been suggested to act as a gatekeeper of colorectal carcinogenesis, which means that functional loss of APC is very closely correlated with the future progression towards malignancy. Around 85% of all sporadic and hereditary colorectal tumors show loss of APC function. In addition to their role in hereditary and somatic CRC, mutations in APC have also been demonstrated in other cancer types, such as Desmoid tumor (aggressive fibromatoses) (11), bladder cancer (12), gastric cancer (13), and breast cancer (14).
APC is a large (312 kDa) protein that has many well-characterized functional domains and interacts with numerous other proteins. However, its critical role in tumorigenesis appears to lie in the control of cellular levels of β-catenin, thus acting as a negative regulator of the Wnt signaling pathway. When APC is mutated, the effector protein of the Wnt signaling pathway, β-catenin, accumulates and translocates into the nucleus. Once there it binds to the Tcf/LEF transcription factors and initiates transcription of a wide variety of genes. The downstream transcriptional activation targets of β-catenin include a number of genes involved in the development and progression of colorectal carcinoma, including Cyclin D1 and the oncogene c-myc. In addition to its role in the Wnt signaling pathway, loss of APC function results in disrupted cell-cell adhesion in cancer cells lacking APC.
Almost all (95%) APC mutations in CRC are nonsense or frameshift mutations that result in a truncated protein product with abnormal function. The sites of the APC mutations are usually not random; there are well-characterized hotspots for the APC-truncating mutations.
As in CRC, a large number of other human genetic diseases result from mutations that cause the premature termination of the synthesis of the protein encoded by the mutant gene, and one way of treating these diseases would be to supplement the mutant gene with a wild-type copy.
For some years it has been known that aminoglycoside antibiotics can suppress disease-associated premature stop mutations by allowing an amino acid to be incorporated in place of a stop codon, thus permitting translation to continue to the normal end of the transcript. Recent studies have shown that aminoglycosides can suppress premature stop mutations in mammalian transcripts both in vitro and in vivo to levels that restore physiologically relevant amounts of functional protein (1-5). The utility of this approach was previously demonstrated with the autosomal recessive disease cystic fibrosis (CF), and in Duchenne muscular dystrophy (DMD) patients with nonsense mutations. In both CF and DMD patients, only 5 to 10% of all cases are due to a nonsense mutation in the coding sequence. In comparison, in patients with colorectal tumors, premature stop codons in the APC gene are found in around 85% of all cases (6). However, studies aimed at restoring the full-length APC protein into cells that lack a functional APC protein have not been conducted so far.
Aminoglycosides such as gentamicin have serious dose-limiting toxicities and require intravenous administration, thus making them an unattractive long-term treatment for cancers.
The approach of suppressing stop codon mutations may be beneficial to other patients suffering from diseases that result from a stop codon mutation in an important gene (for example in patients suffering from APTR deficiency, antithromnin III, acid spingumyelias, and Hailey-Hailey disease, reviewed in (8)).
Recently, it has been shown that antibiotics of the macrolide family can target the ribosomal 50S subunit and induce stop codon readthrough in a prokaryotic system (7). U.S. Pat. No. 5,324,720 and U.S. Pat. Appl. Pub. No. 2005/0171032 relate to methods of treating cancer using certain macrolide antibiotics (9, 10).